Project Summary/Abstract Understanding how large macromolecules are transported across a cell wall is a complex and poorly understood biological process. The nosocomial pathogen Clostridioides difficile produces and secretes two large toxins that are responsible for causing disease. Although much is known about the effector functions of the toxins, very little is known about how the toxins are secreted. The toxins are encoded on a pathogenicity locus which also encodes TcdE, a holin-like protein and TcdL, the N-terminal remnant of an endolysin. While bacteriophages use holin/endolysin systems to trigger bacterial cell lysis and escape, multiple reports now suggest that TcdE is used for the secretion of the toxins. This proposal will address the outstanding questions of how TcdE and TcdL interact with C. difficile toxins to create a pore and secrete toxins without causing cell lysis. TcdE and TcdL will be fluorescently tagged, and their localization and oligomerization will be determined in vivo using structured illumination microscopy and stepwise photobleaching. The oligomerization state(s) of TcdE will also be determined in vitro using cryo-transmission electron microscopy (cryo-TEM). TcdL has recently been discovered and its role in toxin secretion has not been fully explored. TcdL will be deleted and overexpressed to determine what, if any, role it has during toxin secretion. Direct binding assays will assess if TcdL can interact with the toxins or TcdE to facilitate toxin secretion. Finally, the cell wall architecture of C. difficile will be assessed during toxin secretion using correlative light and electron microscopy, and focused ion-beam scanning electron microscopy. The 3D structure of TcdE and the cell wall will be determined in situ using cryo-electron tomography. This proposal will shed new light on a basic biological process: the transport of macromolecules across the cell membrane. The experiments proposed here will build upon my strong foundation in bacterial genetics and physiology. I will be training in the laboratory of Dr. D. Borden Lacy, an established leader in the field of structural biology with a specialty in solving high-resolution toxin structures. Her guidance will allow me to learn cutting edge techniques in structural biology and high-resolution light microscopy. The techniques that I will learn and the research program that I will build over the course of this training will allow me to transition into an independent investigator at a R1 research institution.